Methods and compositions for treating and preventing disease associated with alpha-V beta-8 integrin

ABSTRACT

Antibodies (e.g., isolated antibodies) that specifically bind to human integrin β8 and inhibit adhesion of latency associated peptide (LAP) to αvβ8 are provided. In some embodiments, the antibody cross-reacts with mouse integrin β8. In some embodiments, the antibody blocks TGFJ3 activation. In some embodiments, the antibody antagonizes binding of LAP to αβ8 with an IC50 below 5 nM.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is the US National Stage entry of International Appl.No. PCT/US2014/032550, filed Apr. 1, 2014, which claims priority to U.S.Provisional Appl. No. 61/807,195, filed Apr. 1, 2013, the disclosures ofeach are incorporated herein in their entireties.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant nos. R37HL053949 and U19 AI077439 awarded by the National Institutes of Health.The government has certain rights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 081906-0958641_ST25.txt, created onSep. 21, 2015, 46,282 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Members of the integrin family recognize a variety ofspatially-restricted extracellular ligands. Classically, ligation ofintegrins activates cytoplasmic signals in the integrin-expressing celland contributes to cell adhesion, migration, proliferation and survival.At least two members of this family, αvβ6 and αvβ8, perform anadditional function, activation of latent complexes of transforminggrowth factor β. In effect, this process allows integrins on one cell toactivate signals on adjacent (in the case of αvβ6) or nearby cells (inthe case of αvβ8). Integrin-mediated TGFβ activation has been shown toplay roles in, for example, modulating tissue fibrosis, acute lunginjury and pulmonary emphysema.

BRIEF SUMMARY OF THE INVENTION

Antibodies (e.g., isolated antibodies) that specifically bind to humanintegrin β8 and inhibit adhesion of latency associated peptide (LAP) toαvβ8 are provided. In some embodiments, the antibody cross-reacts withmouse integrin β8. In some embodiments, the antibody blocks TGFβactivation. In some embodiments, the antibody antagonizes binding of LAPto αvβ8 with an IC₅₀ below 5 nM.

In some embodiments, the antibody competes for binding with an antibodyselected from the group consisting of ADWA-2 (ADWA-2, ADWA-2-1 andADWA-2-2), ADWA-8 (ADWA-8, ADWA-8-1, ADWA-8-2, ADWA-8-3), ADWA-10,ADWA-11, ADWA-13 (ADWA-13, ADWA-13-1, ADWA-13-2), ADWA-15, ADWA-16,ADWA-25, and ADWA-20.

In some embodiments, the antibody comprises the complementaritydetermining regions (CDR1, CDR2, and CDR3) of the heavy and light chainvariable regions of an antibody selected from ADWA-2 (ADWA-2, ADWA-2-1and ADWA-2-2), ADWA-8 (ADWA-8, ADWA-8-1, ADWA-8-2, ADWA-8-3), ADWA-10,ADWA-11, ADWA-13 (ADWA-13, ADWA-13-1, ADWA-13-2), ADWA-15, ADWA-16,ADWA-25, and ADWA-20. In some embodiments, the antibody comprises theChothia-determined CDR1, CDR2, and CDR3 of the heavy and light chainvariable regions of an antibody selected from ADWA-2 (ADWA-2, ADWA-2-1and ADWA-2-2), ADWA-8 (ADWA-8, ADWA-8-1, ADWA-8-2, ADWA-8-3), ADWA-10,ADWA-11, ADWA-13 (ADWA-13, ADWA-13-1, ADWA-13-2), ADWA-15, ADWA-16,ADWA-25, and ADWA-20. In some embodiments, antibody comprises theKabat-determined CDR1, CDR2, and CDR3 of the heavy and light chainvariable regions of an antibody selected from ADWA-2 (ADWA-2, ADWA-2-1and ADWA-2-2), ADWA-8 (ADWA-8, ADWA-8-1, ADWA-8-2, ADWA-8-3), ADWA-10,ADWA-11, ADWA-13 (ADWA-13, ADWA-13-1, ADWA-13-2), ADWA-15, ADWA-16,ADWA-25, and ADWA-20.

In some embodiments, the antibody comprises the heavy and light chainvariable regions of an antibody selected from ADWA-2 (ADWA-2, ADWA-2-1and ADWA-2-2), ADWA-8 (ADWA-8, ADWA-8-1, ADWA-8-2, ADWA-8-3), ADWA-10,ADWA-11, ADWA-13 (ADWA-13, ADWA-13-1, ADWA-13-2), ADWA-15, ADWA-16,ADWA-25, and ADWA-20.

In some embodiments, the antibody comprises the heavy chain CDRs(Chothia or Kabat) shown in SEQ ID NOs:23-25, and the light chain CDRsshown in SEQ ID NOs:46-48. In some embodiments, the antibody comprisesthe heavy chain CDRs (Chothia or Kabat) shown in SEQ ID NOs:23, 26, and25, and the light chain CDRs shown in SEQ ID NOs:46-48. In someembodiments, the antibody comprises the heavy chain CDRs (Chothia orKabat) shown in SEQ ID NOs:27-29, and the light chain CDRs shown in SEQID NOs:46, 49, and 50. In some embodiments, the antibody comprises theheavy chain CDRs (Chothia or Kabat) shown in SEQ ID NOs:30-32, and thelight chain CDRs shown in SEQ ID NOs:51-53. In some embodiments, theantibody comprises the heavy chain CDRs (Chothia or Kabat) shown in SEQID NOs:33-35, and the light chain CDRs shown in SEQ ID NOs:54-56. Insome embodiments, the antibody comprises the heavy chain CDRs (Chothiaor Kabat) shown in SEQ ID NOs:36-38, and the light chain CDRs shown inSEQ ID NOs:57-59. In some embodiments, the antibody comprises the heavychain CDRs (Chothia or Kabat) shown in SEQ ID NOs:36, 39, and 38, andthe light chain CDRs shown in SEQ ID NOs:57-59. In some embodiments, theantibody comprises the heavy chain CDRs (Chothia or Kabat) shown in SEQID NOs:40-42, and the light chain CDRs shown in SEQ ID NOs:60-62. Insome embodiments, the antibody comprises the heavy chain CDRs (Chothiaor Kabat) shown in SEQ ID NOs:43-45, and the light chain CDRs shown inSEQ ID NOs:63-65.

Any of the antibodies described herein can include one or more humanframework region (e.g., 1, 2, 3, or 4 FRs). In some embodiments, the oneor more human framework region includes at least one back mutation.Pharmaceutical compositions comprising any of the antibodies describedherein are also provided.

Isolated nucleic acid encoding any of the antibodies described hereinare also provided (e.g., SEQ ID NOs:66-87, and conservative variantsthereof). Expression vectors comprising the nucleic acid are alsoprovided. Isolated host cells comprising the vectors are also provided.

Also provided are methods of reducing TGFβ activation in a human in needthereof. In some embodiments, the methods comprise administering asufficient amount of the antibody as described herein to the human,thereby reducing TGFβ activation in the human. In some embodiments, thehuman has a disease selected from the group consisting of asthma,multiple sclerosis or acute lung injury and at least one symptom of thedisease is ameliorated by the reduced TGFβ activation. In someembodiments, the human has a disease selected from the group consistingof rheumatoid arthritis, psoriasis and chronic obstructive pulmonarydisease and at least one symptom of the disease is ameliorated by thereduced TGFβ activation.

Also provided is a method of detecting the presence or quantity ofintegrin β8 in a biological sample. In some embodiments, the methodcomprises contacting the biological sample with the antibody asdescribed herein and detecting the presence, absence, or quantity of theantibody specifically bound to β8 in the sample. In some embodiments,the antibody is linked to a detectable label, wherein the label isfluorescent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides flow cytometry plots of primary astrocytes bound byantibodies as described in Example 1.

FIG. 2 shows data from LAP adhesion blocking experiments as described inExample 1.

FIG. 3 shows data for TGFβ activation as described in Example 1.

FIG. 4 shows data for ADWA-2 blocking of TGFβ activation at theindicated concentrations. Controls shown on the right include the 1D11antibody specific for TGFβ, and no antibody.

FIG. 5 shows data for ADWA-11 blocking of TGFβ activation at theindicated concentrations. Controls shown on the right include the 1D11antibody specific for TGFβ, and no antibody.

FIG. 6 shows data for ADWA-13 blocking of TGFβ activation at theindicated concentrations. Controls shown on the right include the 1D11antibody specific for TGFβ, and no antibody.

FIG. 7 shows data for ADWA-15 blocking of TGFβ activation at theindicated concentrations. Controls shown on the right include the 1D11antibody specific for TGFβ, and no antibody.

FIG. 8 shows data for ADWA-16 blocking of TGFβ activation at theindicated concentrations. Controls shown on the right include the 1D11antibody specific for TGFβ, and no antibody.

FIG. 9 shows data for ADWA-11 blocking of LAP adhesion. LAPconcentrations for each sample are, from left to right, 3 ug/ml, 1ug/ml, and 0.3 ug/ml. Controls are no antibody (left) and 3G9 antibodyspecific for 06 (middle).

FIG. 10 shows data for ADWA-2 (left) and ADWA-16 (right) blocking of LAPadhesion at the indicated concentrations. LAP was contacted with cellsat 1 ug/ml.

FIG. 11 shows data for ADWA-11 blocking of LAP adhesion. LAPconcentrations for each sample are shown. Controls are no antibody(left) and 3G9 antibody specific for β6 (middle) for each concentrationof LAP.

FIG. 12 shows data for ADWA-2, ADWA-8, ADWA-10, ADWA-11, and ADWA-13blocking of LAP adhesion at various antibody concentrations. LAP wascontacted with cells at 1 ug/ml. For each antibody, left to right,concentration was 10 ug/ml, 1 ug/ml, and 0.1 ug/ml. Results for control3G9 antibody are shown on the right.

FIG. 13 shows data for ADWA-15, ADWA-16, ADWA-20, and ADWA-25 blockingof LAP adhesion at various antibody concentrations. LAP was contactedwith cells at 1 ug/ml. For each antibody, left to right, concentrationwas 10 ug/ml, 1 ug/ml, and 0.1 ug/ml. Results for control 3G9 antibodyare shown on the right.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Various antibodies that bind to human integrin β8 and that inhibitadhesion of latency associated peptide (LAP) are provided.

II. Definitions

An “antagonist” refers to an agent that binds to an integrin (e.g.,αvβ8) and partially or totally blocks stimulation, decreases, prevents,delays activation, inactivates, desensitizes, or down regulates theactivity of the integrin.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site ncbi.nlm.nih.gov/BLAST/ or the like). Such sequencesare then said to be “substantially identical.” The present inventionprovides for, e.g., antibodies having polynucleotide or polypeptidesequences that have at least 80% identity, e.g., 85%, 90%, 91%, 92%, 93,94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, to a reference sequence,e.g., CDRs or variable regions of any of antibodies ADWA-2, ADWA-8,ADWA-10, ADWA-11, ADWA-13, ADWA-15, ADWA-16, ADWA-25, and ADWA-20. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 or more amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

An algorithm that is suitable for determining percent sequence identityand sequence similarity are the BLAST and BLAST 2.0 algorithms, whichare described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977)and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively.BLAST and BLAST 2.0 are used, with the parameters described herein, todetermine percent sequence identity for the nucleic acids and proteinsof the invention. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms encompass to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

An antibody as described herein can consist of one or more polypeptidessubstantially encoded by immunoglobulin genes or fragments ofimmunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Insome embodiments, the antibody is IgG (e.g., IgG1, IgG2, IgG3, IgG4),IgM, IgA, IgD, or IgE.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

The term “antibody” as used herein includes antibody fragments thatretain binding specificity. For example, there are a number of wellcharacterized antibody fragments. Thus, for example, pepsin digests anantibody C-terminal to the disulfide linkages in the hinge region toproduce F(ab)′2, a dimer of Fab which itself is a light chain joined toVH-CH1 by a disulfide bond. The F(ab)′2 may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the (Fab′)2 dimer into an Fab′ monomer. The Fab′ monomer isessentially an Fab with part of the hinge region (see, FundamentalImmunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a moredetailed description of other antibody fragments). While variousantibody fragments are defined in terms of the digestion of an intactantibody, one of skill will appreciate that fragments can be synthesizedde novo either chemically or by utilizing recombinant DNA methodology.Thus, the term antibody, as used herein also includes antibody fragmentseither produced by the modification of whole antibodies or synthesizedusing recombinant DNA methodologies.

In an antibody, substitution variants have at least one amino acidresidue removed and a different residue inserted in its place. The sitesof greatest interest for substitutional mutagenesis include thehypervariable regions, but framework alterations are also contemplated.Examples of conservative substitutions are described above.

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a β-sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) Polar without charge: Cys, Ser, Thr, Asn, Gln;    -   (3) Acidic (negatively charged): Asp, Glu;    -   (4) Basic (positively charged): Lys, Arg;    -   (5) Residues that influence chain orientation: Gly, Pro; and    -   (6) Aromatic: Trp, Tyr, Phe, His.        Non-conservative substitutions are made by exchanging a member        of one of these classes for another class.

One type of substitution that can be made is to change one or morecysteines in the antibody, which may be chemically reactive, to anotherresidue, such as, without limitation, alanine or serine. For example,there can be a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant region of an antibody. In some embodiments,the cysteine is canonical (e.g., involved in disulfide bond formation).Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross-linking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is an antibodyfragment such as an Fv fragment.

Antibodies include V_(H)-V_(L) dimers, including single chain antibodies(antibodies that exist as a single polypeptide chain), such as singlechain Fv antibodies (sFv or scFv) in which a variable heavy and avariable light region are joined together (directly or through a peptidelinker) to form a continuous polypeptide. The single chain Fv antibodyis a covalently linked V_(H)-V_(L) which may be expressed from a nucleicacid including V_(H)- and V_(L)-encoding sequences either joineddirectly or joined by a peptide-encoding linker (e.g., Huston, et al.Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). While the V_(H) andV_(L) are connected to each as a single polypeptide chain, the V_(H) andV_(L) domains associate non-covalently. Alternatively, the antibody canbe another fragment. Other fragments can also be generated, e.g., usingrecombinant techniques, as soluble proteins or as fragments obtainedfrom display methods. Antibodies can also include diantibodies andminiantibodies. Antibodies of the invention also include heavy chaindimers, such as antibodies from camelids. For the purposes of thisinventor, antibodies are employed in a form that can activate EphA3present on the surface of myeloproliferative cells or that can killmyeloproliferative cells by ADCC. Thus, in some embodiments an antibodyis dimeric. In other embodiments, the antibody may be in a monomericform that has an active isotype. In some embodiments the antibody is ina multivalent form, e.g., a trivalent or tetravalent form, that cancross-link EphA3.

As used herein, “V-region” refers to an antibody variable region domaincomprising the segments of Framework (FW) 1, complementarity-determiningregion (CDR) 1, FW2, CDR2, FW3, CDR3, and Framework 4. The V region forthe heavy and light chains is commonly designated V_(H) and V_(L),respectively, or with like terms. The V region is included on Fab,F(ab′)2, Fv and scFv antibody fragments described herein, and involvedin specific antigen recognition.

As used herein, “complementarity-determining region (CDR)” refers to thethree hypervariable regions in each chain that interrupt the four“framework (FW)” regions established by the light and heavy chainvariable regions. The CDRs are primarily responsible for binding to anepitope of an antigen. The CDRs of each chain are typically referred toas CDR1, CDR2, and CDR3, numbered sequentially starting from theN-terminus, and are also typically identified by the chain in which theparticular CDR is located. Thus, a V_(H) CDR3 is located in the variabledomain of the heavy chain of the antibody in which it is found, whereasa V_(L) CDR1 is the CDR1 from the variable domain of the light chain ofthe antibody in which it is found.

The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The amino acid sequences of the CDRs and framework regions can bedetermined using various well known definitions in the art, e.g., Kabat,Chothia, international ImMunoGeneTics database (IMGT), and AbM (see,e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structuresfor the hypervariable regions of immunoglobulins. J. Mol. Biol. 196,901-917; Chothia C. et al., 1989, Conformations of immunoglobulinhypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992,structural repertoire of the human VH segments J. Mol. Biol. 227,799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). Definitions ofantigen combining sites are also described in the following: Ruiz etal., IMGT, the international ImMunoGeneTics database. Nucleic AcidsRes., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the internationalImMunoGeneTics database. Nucleic Acids Res. January 1; 29(1):207-9(2001); MacCallum et al, Antibody-antigen interactions: Contact analysisand binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); andMartin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin,et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al,Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M. J. E.(ed.), Protein Structure Prediction. Oxford University Press, Oxford,141-172 1996).

As used herein, “chimeric antibody” refers to an immunoglobulin moleculein which (a) the constant region, or a portion thereof, is altered,replaced or exchanged so that the antigen binding site (variable region)is linked to a constant region of a different or altered class, effectorfunction and/or species, or an entirely different molecule which confersnew properties to the chimeric antibody, e.g., an enzyme, toxin,hormone, growth factor, drug, etc.; or (b) the variable region, or aportion thereof, is altered, replaced or exchanged with a variableregion, or portion thereof, having a different or altered antigenspecificity; or with corresponding sequences from another species orfrom another antibody class or subclass.

As used herein, “humanized antibody” refers to an immunoglobulinmolecule in CDRs from a donor antibody are grafted onto human frameworksequences. Humanized antibodies may also comprise residues of donororigin in the framework sequences. The humanized antibody can alsocomprise at least a portion of a human immunoglobulin constant region.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. Humanization can be performed using methods known in the art(e.g., Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988);Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; U.S. Pat. No.4,816,567), including techniques such as “superhumanizing” antibodies(Tan et al., J. Immunol. 169: 1119, 2002) and “resurfacing” (e.g.,Staelens et al., Mol. Immunol. 43: 1243, 2006; and Roguska et al., Proc.Natl. Acad. Sci USA 91: 969, 1994).

The terms “antigen,” “immunogen,” “antibody target,” “target analyte,”and like terms are used herein to refer to a molecule, compound, orcomplex that is recognized by an antibody, i.e., can be specificallybound by the antibody. The term can refer to any molecule that can bespecifically recognized by an antibody, e.g., a polypeptide,polynucleotide, carbohydrate, lipid, chemical moiety, or combinationsthereof (e.g., phosphorylated or glycosylated polypeptides, etc.). Oneof skill will understand that the term does not indicate that themolecule is immunogenic in every context, but simply indicates that itcan be targeted by an antibody.

Antibodies bind to an “epitope” on an antigen. The epitope is thelocalized site on the antigen that is recognized and bound by theantibody. Epitopes can include a few amino acids or portions of a fewamino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, orportions of those amino acids. In some cases, the epitope includesnon-protein components, e.g., from a carbohydrate, nucleic acid, orlipid. In some cases, the epitope is a three-dimensional moiety. Thus,for example, where the target is a protein, the epitope can be comprisedof consecutive amino acids, or amino acids from different parts of theprotein that are brought into proximity by protein folding (e.g., adiscontinuous epitope). The same is true for other types of targetmolecules that form three-dimensional structures. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed(1996).

A “label” or a “detectable moiety” is a diagnostic agent or componentdetectable by spectroscopic, radiological, photochemical, biochemical,immunochemical, chemical, or other physical means. Exemplary labelsinclude radiolabels (e.g., ¹¹¹In, ^(99m)Tc, ¹³¹I, ⁶⁷Ga) and otherFDA-approved imaging agents. Additional labels include ³²P, fluorescentdyes, electron-dense reagents, enzymes, biotin, digoxigenin, or haptensand proteins or other entities which can be made detectable, e.g., byincorporating a radiolabel into the targeting agent. Any method known inthe art for conjugating a nucleic acid or nanocarrier to the label maybe employed, e.g., using methods described in Hermanson, BioconjugateTechniques 1996, Academic Press, Inc., San Diego.

A “labeled” or “tagged” antibody or agent is one that is bound, eithercovalently, through a linker or a chemical bond, or noncovalently,through ionic, van der Waals, electrostatic, or hydrogen bonds to alabel such that the presence of the antibody or agent may be detected bydetecting the presence of the label bound to the antibody or agent.

Techniques for conjugating detectable and therapeutic agents toantibodies are well known (see, e.g., Arnon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery” in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.),pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982)).

The terms “specific for,” “specifically binds,” and like terms refer toa molecule (e.g., antibody or antibody fragment) that binds to a targetwith at least 2-fold greater affinity than non-target compounds, e.g.,at least any of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 25-fold, 50-fold, or 100-fold greater affinity. For example, anantibody that specifically binds a primary antibody will typically bindthe primary antibody with at least a 2-fold greater affinity than anon-primary antibody target (e.g., an antibody from a different speciesor of a different isotype, or a non-antibody target). Specificity can bedetermined using standard methods, e.g., solid-phase ELISA immunoassays(see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998)for a description of immunoassay formats and conditions that can be usedto determine specific immunoreactivity).

The term “binds” with respect to an antibody target (e.g., antigen,analyte, immune complex), typically indicates that an antibody binds amajority of the antibody targets in a pure population (assumingappropriate molar ratios). For example, an antibody that binds a givenantibody target typically binds to at least ⅔ of the antibody targets ina solution (e.g., at least any of 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100%). One of skill will recognize that somevariability will arise depending on the method and/or threshold ofdetermining binding.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a known compound (positive control). Acontrol can also represent an average value or a range gathered from anumber of tests or results. One of skill in the art will recognize thatcontrols can be designed for assessment of any number of parameters. Forexample, a control can be devised to compare therapeutic benefit basedon pharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of benefit and/or side effects). Controls can be designed forin vitro applications. One of skill in the art will understand whichcontrols are valuable in a given situation and be able to analyze databased on comparisons to control values. Controls are also valuable fordetermining the significance of data. For example, if values for a givenparameter are widely variant in controls, variation in test samples willnot be considered as significant.

The terms “therapeutically effective dose,” “effective dose,” or“therapeutically effective amount” herein is meant a dose that produceseffects for which it is administered. The exact dose and formulationwill depend on the purpose of the treatment, and will be ascertainableby one skilled in the art using known techniques (see, e.g., Lieberman,Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Scienceand Technology of Pharmaceutical Compounding (1999); Remington: TheScience and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003),and Pickar, Dosage Calculations (1999)). For example, for the givenparameter, a therapeutically effective amount will show an increase ordecrease of therapeutic effect at least any of 5%, 10%, 15%, 20%, 25%,40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy canalso be expressed as “-fold” increase or decrease. For example, atherapeutically effective amount can have at least any of a 1.2-fold,1.5-fold, 2-fold, 5-fold, or more effect over a control.

The term “pharmaceutically acceptable salts” or “pharmaceuticallyacceptable carrier” is meant to include salts of the active compoundswhich are prepared with relatively nontoxic acids or bases, depending onthe particular substituents found on the antibodies described herein.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable acid addition salts includethose derived from inorganic acids like hydrochloric, hydrobromic,nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19(1977)). Certain specific compounds of the present invention containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts. Otherpharmaceutically acceptable carriers known to those of skill in the artare suitable for the present invention.

The term “reduce,” “reducing,” or “reduction,” when used in the contextof αvβ8-mediate TGFβ activation refers to any detectable negative changeor decrease in quantity of a parameter that reflects TGFβ activation,compared to a standard value obtained under the same conditions but inthe absence of an antibody as described herein (e.g., anti-αvβ8antibodies). The level of this decrease following exposure to anantibody as described herein (e.g., anti-αvβ8 antagonists, anti-αvβ8antibodies and immunoconjugates) is, in some embodiments, at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen-binding portion thereof, competesfor binding with a second antibody, or an antigen-binding portionthereof, where binding of the first antibody with its cognate epitope isdetectably decreased in the presence of the second antibody compared tothe binding of the first antibody in the absence of the second antibody.The alternative, where the binding of the second antibody to its epitopeis also detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its cognate epitope or ligand, whether to the same,greater, or lesser extent, the antibodies are said to “cross-compete”with each other for binding of their respective epitope(s). Bothcompeting and cross-competing antibodies are encompassed by the presentinvention. Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof, and the like), theskilled artisan would appreciate, based upon the teachings providedherein, that such competing and/or cross-competing antibodies areencompassed and can be useful for the methods disclosed herein.

Numerous types of competitive binding assays are known, for example:solid phase direct or indirect radioimmunoassay (RIA), solid phasedirect or indirect enzyme immunoassay (EIA), sandwich competition assay(see Stahli et al., Methods in Enzymology 9:242-253 (1983)); solid phasedirect biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, Antibodies, A Laboratory Manual,Cold Spring Harbor Press (1988)); solid phase direct label RIA usingI-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabelled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50 or 75%.

III. Antibodies that Bind Integrin β8

Antibodies (including antibody fragments) that specifically bind tohuman integrin β8 are provided, as well as methods for treating orpreventing diseases for which decrease of TGFβ activation has anameliorative effect. “Integrin β8” is used interchangeably with β8 andbeta-8. The human integrin β8 protein sequence can be found at Uniprotaccession number P26012, while the murine integrin β8 sequence hasUniprot accession number QOVBD0. See, also, Moyle et al. Journal ofBiological Chemistry 266:19650-19658 (1991); Nishimura et al., J.Biological Chemistry 269:28708-28715 (1994).

In some embodiments, an antibody that specifically binds to humanintegrin β8 and inhibits (partially or completely blocks) binding oflatency associated peptide (LAP) to αvβ8 is provided. LAP is a ligandfor αvβ8. See, e.g., Sheppard, Cancer and Metastasis Reviews24(3):395-402 (2005); Lu et al. J Cell Sci 115:4641-4648 (2002). Asshown in FIG. 2, antibodies described in the Example inhibit biding ofLAP to αvβ8. Antibodies can antagonize LAP binding to αvβ8 with an IC₅₀of, for example, less than e.g., 10, 5, 1, 0.1 nM or lower.

In some embodiments, an antibody of the invention specifically binds tomouse as well as human integrin β8. Each of antibodies ADWA-2, ADWA-8,ADWA-10, ADWA-11, ADWA-13, ADWA-15, ADWA-16, ADWA-25, and ADWA-20 bindto both human and mouse β8. One advantage of such antibodies is thatclinical data can be generated for these antibodies in mice as well ashumans.

One aspect of blockage of LAP binding to αvβ8 in a cell can be that theantibodies prevent or reduce TGFβ activate in the cell. Thus, in someembodiments, the antibodies described herein are useful for decreasingTGFβ activation in a cell or an animal (e.g., a mouse, human, or otheranimal).

In some embodiments, the antibodies compete with one of more antibodiesselected from ADWA-2, ADWA-8, ADWA-10, ADWA-11, ADWA-13, ADWA-15,ADWA-16, ADWA-25, and ADWA-20. In some embodiments, the antibodies bindto the same epitope as bound by ADWA-2, ADWA-8, ADWA-10, ADWA-11,ADWA-13, ADWA-15, ADWA-16, ADWA-25, and ADWA-20. Monoclonal antibodies,and chimeric, and especially humanized antibodies, are of particular usefor human therapeutic uses of the antibodies described herein. Thus, insome embodiments, a αvβ8-specific antibody as described herein comprisesthe complementarity determining regions (CDRs) of the heavy chainvariable region and the light chain variable region of an antibodyselected from ADWA-2, ADWA-8, ADWA-10, ADWA-11, ADWA-13, ADWA-15,ADWA-16, ADWA-25, and ADWA-20. For example, the antibody can have CDR1,CDR2, and CDR3 of the heavy chain variable region and CDR1, CDR2, andCDR3 of the light chain variable region of an antibody selected fromADWA-2, ADWA-8, ADWA-10, ADWA-11, ADWA-13, ADWA-15, ADWA-16, ADWA-25,and ADWA-20 and have a heterologous (e.g., human or human-like)framework region.

In some embodiments, the αvβ8-specific antibodies as described hereincomprise the complementarity determining regions (CDRs) of the heavychain variable region sequence and the light chain variable regionsequence selected from:

-   -   SEQ ID NO:1 and SEQ ID NO:16, respectively;    -   SEQ ID NO:2 and SEQ ID NO:16, respectively;    -   SEQ ID NO:3 and SEQ ID NO:16, respectively;    -   SEQ ID NO:4 and SEQ ID NO:16, respectively;    -   SEQ ID NO:5 and SEQ ID NO:16, respectively;    -   SEQ ID NO:6 and SEQ ID NO:16, respectively;    -   SEQ ID NO:7 and SEQ ID NO:17, respectively;    -   SEQ ID NO:8 and SEQ ID NO:18, respectively;    -   SEQ ID NO:9 and SEQ ID NO:19, respectively;    -   SEQ ID NO:10 and SEQ ID NO:20, respectively;    -   SEQ ID NO:11 and SEQ ID NO:20, respectively;    -   SEQ ID NO:12 and SEQ ID NO:20, respectively;    -   SEQ ID NO:13 and SEQ ID NO:20, respectively;    -   SEQ ID NO:14 and SEQ ID NO:21, respectively; and    -   SEQ ID NO:15 and SEQ ID NO:22, respectively.

In some embodiments, the αvβ8-specific antibodies as described hereincomprise the heavy chain variable region sequence and the light chainvariable region sequence of selected from:

-   -   SEQ ID NO:1 and SEQ ID NO:16, respectively;    -   SEQ ID NO:2 and SEQ ID NO:16, respectively;    -   SEQ ID NO:3 and SEQ ID NO:16, respectively;    -   SEQ ID NO:4 and SEQ ID NO:16, respectively;    -   SEQ ID NO:5 and SEQ ID NO:16, respectively;    -   SEQ ID NO:6 and SEQ ID NO:16, respectively;    -   SEQ ID NO:7 and SEQ ID NO:17, respectively;    -   SEQ ID NO:8 and SEQ ID NO:18, respectively;    -   SEQ ID NO:9 and SEQ ID NO:19, respectively;    -   SEQ ID NO:10 and SEQ ID NO:20, respectively;    -   SEQ ID NO:11 and SEQ ID NO:20, respectively;    -   SEQ ID NO:12 and SEQ ID NO:20, respectively;    -   SEQ ID NO:13 and SEQ ID NO:20, respectively;    -   SEQ ID NO:14 and SEQ ID NO:21, respectively; and    -   SEQ ID NO:15 and SEQ ID NO:22, respectively.

In some embodiments, the antibody has the heavy chain CDR sequences andlight chain CDR sequences found in SEQ ID NO:1 and SEQ ID NO:16,respectively. In some embodiments, the antibody has the heavy chain CDRsequences and light chain CDR sequences found in SEQ ID NO:4 and SEQ IDNO:16, respectively. In some embodiments, the antibody has the heavychain CDR sequences and light chain CDR sequences found in SEQ ID NO:7and SEQ ID NO:17, respectively. In some embodiments, the antibody hasthe heavy chain CDR sequences and light chain CDR sequences found in SEQID NO:8 and SEQ ID NO:18, respectively. In some embodiments, theantibody has the heavy chain CDR sequences and light chain CDR sequencesfound in SEQ ID NO:9 and SEQ ID NO:19, respectively.

Monoclonal antibodies can be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, for example, Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246: 1275-1281 (1989).

Monoclonal antibodies can be collected and titered against a β8 ligand(e.g., LAP) in an immunoassay, for example, a solid phase immunoassaywith the ligand immobilized on a solid support. In some embodiments,monoclonal antibodies will bind with a K_(d) of at least about 0.1 mM,e.g., at least about 1 μM, e.g., at least about 0.1 μM or better, e.g.,0.01 μM or lower.

In an exemplary embodiment, an animal, such as a rabbit or mouse isimmunized with a β8 polypeptide, or an nucleic acid construct encodingsuch a polypeptide. The antibodies produced as a result of theimmunization can be isolated using standard methods. In someembodiments, the animal is a knockout of integrin β8 and is immunizedwith a human β8 integrin polypeptide or a fragment thereof.

The immunoglobulins, including binding fragments and other derivativesthereof, of the present invention may be produced readily by a varietyof recombinant DNA techniques, including by expression in transfectedcells (e.g., immortalized eukaryotic cells, such as myeloma or hybridomacells) or in mice, rats, rabbits, or other vertebrate capable ofproducing antibodies by well-known methods. Suitable source cells forthe DNA sequences and host cells for immunoglobulin expression andsecretion can be obtained from a number of sources, such as the AmericanType Culture Collection (Catalogue of Cell Lines and Hybridomas, Fifthedition (1985) Rockville, Md.).

In some embodiments, the antibody is a humanized antibody, i.e., anantibody that retains the reactivity of a non-human antibody while beingless immunogenic in humans. This can be achieved, for instance, byretaining the non-human CDR regions and replacing the remaining parts ofthe antibody with their human counterparts. See, e.g., Morrison et al.,PNAS USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92(1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec.Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994).Techniques for humanizing antibodies are well known in the art and aredescribed in e.g., U.S. Pat. Nos. 4,816,567; 5,530,101; 5,859,205;5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and6,329,511; WO 87/02671; EP Patent Application 0173494; Jones et al.(1986) Nature 321:522; and Verhoyen et al. (1988) Science 239:1534.Humanized antibodies are further described in, e.g., Winter and Milstein(1991) Nature 349:293. For example, polynucleotides comprising a firstsequence coding for humanized immunoglobulin framework regions and asecond sequence set coding for the desired immunoglobulincomplementarity determining regions can be produced synthetically or bycombining appropriate cDNA and genomic DNA segments. Human constantregion DNA sequences can be isolated in accordance with well-knownprocedures from a variety of human cells. The CDRs for producing theimmunoglobulins of the present invention will be similarly derived frommonoclonal antibodies capable of specifically binding to αvβ8 integrin(e.g., ADWA-2, ADWA-8, ADWA-10, ADWA-11, ADWA-13, ADWA-15, ADWA-16,ADWA-25, and ADWA-20 or antibodies that compete with ADWA-2, ADWA-8,ADWA-10, ADWA-11, ADWA-13, ADWA-15, ADWA-16, ADWA-25, and ADWA-20 forspecific binding to αvβ8 integrin).

In some cases, transfer of a CDR to a human framework leads to a loss ofspecificity for the humanized antibody. In these cases, back mutationcan be introduced into the framework regions of the human portion of theantibody. Methods of making back mutations are well known in the art andare described in, e.g., Co et al., PNAS USA 88; 2269-2273 (1991) and WO90/07861.

In some embodiments, the antibodies are antibody fragments such as Fab,F(ab′)2, Fv or scFv. The antibody fragments can be generated using anymeans known in the art including, chemical digestion (e.g., papain orpepsin) and recombinant methods. Methods for isolating and preparingrecombinant nucleic acids are known to those skilled in the art (see,Sambrook et al., Molecular Cloning. A Laboratory Manual (2d ed. 1989);Ausubel et al., Current Protocols in Molecular Biology (1995)). Theantibodies can be expressed in a variety of host cells, including E.coli, other bacterial hosts, yeast, and various higher eukaryotic cellssuch as the COS, CHO, and HeLa cells lines and myeloma cell lines.

Competitive binding assays can be used to identify antibodies thatcompete with an antibody described herein for specific binding to αvβ8integrin. Any of a number of competitive binding assays known in the artcan be used to measure competition between two antibodies to the sameantigen. Briefly, the ability of different antibodies to inhibit thebinding of another antibody is tested. For example, antibodies can bedifferentiated by the epitope to which they bind using a sandwich ELISAassay. This is carried out by using a capture antibody to coat thesurface of a well. A subsaturating concentration of tagged-antigen isthen added to the capture surface. This protein will be bound to theantibody through a specific antibody:epitope interaction. After washinga second antibody, which has been covalently linked to a detectablemoiety (e.g., HRP, with the labeled antibody being defined as thedetection antibody) is added to the ELISA. If this antibody recognizesthe same epitope as the capture antibody it will be unable to bind tothe target protein as that particular epitope will no longer beavailable for binding. If however this second antibody recognizes adifferent epitope on the target protein it will be able to bind and thisbinding can be detected by quantifying the level of activity (and henceantibody bound) using a relevant substrate. The background is defined byusing a single antibody as both capture and detection antibody, whereasthe maximal signal can be established by capturing with an antigenspecific antibody and detecting with an antibody to the tag on theantigen. By using the background and maximal signals as references,antibodies can be assessed in a pair-wise manner to determine epitopespecificity.

A first antibody is considered to competitively inhibit binding of asecond antibody, if binding of the second antibody to the antigen isreduced by at least 30%, usually at least about 40%, 50%, 60% or 75%,and often by at least about 90%, in the presence of the first antibodyusing any of the assays described above.

IV. Therapeutic Treatment

As discussed above, the antibodies (including antibody fragments)described herein can be used to reduce TGFβ activation in a cell or ananimal. Accordingly, the antibodies can be administered to an animal(e.g., a human or non-human animal) in need thereof, thereby reducingTGFβ activation in the animal. Diseases for which reduction of TGFβ isat least ameliorative include, but are not limited to, asthma, multiplesclerosis, acute lung injury, rheumatoid arthritis, psoriasis andchronic obstructive pulmonary disease. For example, the inventors havefound that β8 knockout mice have ameliorated symptoms in asthma,multiple sclerosis, and acute lung injury mouse models compared to thosemouse models expressing native integrin β8.

Accordingly, compositions, including pharmaceutical compositions,comprising one or more antibody described herein are provided. Forexample, a pharmaceutical composition comprising a humanized ADWA-2,ADWA-8, ADWA-10, ADWA-11, ADWA-13, ADWA-15, ADWA-16, ADWA-25, or ADWA-20antibody is provided.

In some embodiments, the pharmaceutical composition comprises anantibody or antibody fragment comprising the heavy chain CDR sequences(CDR1, CDR2, and CDR3) and light chain CDR sequences (CDR1, CDR2, andCDR3) selected from the group consisting of:

-   -   SEQ ID NO:1 and SEQ ID NO:16, respectively;    -   SEQ ID NO:2 and SEQ ID NO:16, respectively;    -   SEQ ID NO:3 and SEQ ID NO:16, respectively;    -   SEQ ID NO:4 and SEQ ID NO:16, respectively;    -   SEQ ID NO:5 and SEQ ID NO:16, respectively;    -   SEQ ID NO:6 and SEQ ID NO:16, respectively;    -   SEQ ID NO:7 and SEQ ID NO:17, respectively;    -   SEQ ID NO:8 and SEQ ID NO:18, respectively;    -   SEQ ID NO:9 and SEQ ID NO:19, respectively;    -   SEQ ID NO:10 and SEQ ID NO:20, respectively;    -   SEQ ID NO:11 and SEQ ID NO:20, respectively;    -   SEQ ID NO:12 and SEQ ID NO:20, respectively;    -   SEQ ID NO:13 and SEQ ID NO:20, respectively;    -   SEQ ID NO:14 and SEQ ID NO:21, respectively; and    -   SEQ ID NO:15 and SEQ ID NO:22, respectively.

In some embodiments, the antibody has the heavy chain CDR sequences andlight chain CDR sequences found in SEQ ID NO:1 and SEQ ID NO:16,respectively. In some embodiments, the antibody has the heavy chain CDRsequences and light chain CDR sequences found in SEQ ID NO:4 and SEQ IDNO:16, respectively. In some embodiments, the antibody has the heavychain CDR sequences and light chain CDR sequences found in SEQ ID NO:7and SEQ ID NO:17, respectively. In some embodiments, the antibody hasthe heavy chain CDR sequences and light chain CDR sequences found in SEQID NO:8 and SEQ ID NO:18, respectively. In some embodiments, theantibody has the heavy chain CDR sequences and light chain CDR sequencesfound in SEQ ID NO:9 and SEQ ID NO:19, respectively.

The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there are a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

The compositions of the invention, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, orally, nasally, topically, intravenously,intraperitoneally, or intrathecally. The formulations of compounds canbe presented in unit-dose or multi-dose sealed containers, such asampoules and vials. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.The modulators can also be administered as part a of prepared food ordrug.

Formulations suitable for oral administration can comprise: (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. The optimal dose level for any patient will depend ona variety of factors including the efficacy of the specific modulatoremployed, the age, body weight, physical activity, and diet of thepatient, on a possible combination with other drugs, and on the severityof the PE. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side-effects that accompanythe administration of a particular compound or vector in a particularsubject.

In determining the effective amount of the antagonists of αvβ8 integrinto be administered a physician may evaluate circulating plasma levels ofthe antagonist and antagonist toxicity. In general, the dose equivalentof an antagonist is from about 1 ng/kg to 10 mg/kg for a typicalsubject.

For administration, the antagonists of αvβ8 integrin can be administeredat a rate determined by the LD₅₀ of the antagonist, and the side-effectsof the antagonist at various concentrations, as applied to the mass andoverall health of the subject. Administration can be accomplished viasingle or divided doses.

The compositions may be administered on a regular basis (e.g., daily)for a period of time (e.g., 2, 3, 4, 5, 6, days or 1-3 weeks or more).The compositions can be administered directly to the mammalian subjectto reduce TGFβ activation using any route known in the art, includinge.g., by injection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular, or intrademal), inhalation, transdermal application,rectal administration, or oral administration.

EXAMPLES Example 1

Nine antibodies (designated ADWA-2, ADWA-8, ADWA-10, ADWA-11, ADWA-13,ADWA-15, ADWA-16, ADWA-20, and ADWA-25) were identified that blockadhesion to TGFβ1 LAP, cross react with murine β8 (as determined withflow cytometry on murine astrocytes) and block TGFβ activation (asdetermined with β83.7.12-serial dilutions in a co-culture bioassay withmink lung epithelial cells). The most potent antibody, ADWA11, appearsto have an IC50 for inhibiting TGFβ activation of less than 5 nM (basedon nearly complete inhibition by a concentration of antibody of 1microgram/ml (˜6.3 nM)).

The antibodies were made by immunizing rare β8 knockout mice thatsurvive into young adulthood with purified human αvβ8. Flow cytometryplots are with primary murine astrocytes and show that ADWA-2, ADWA-8,ADWA-10, ADWA-11, ADWA-13, ADWA-15, ADWA-16, ADWA-20, and ADWA-25 allrecognize the mouse integrin, whereas several others do not. See,FIG. 1. The same antibodies block adhesion of 08 transfected SW480 cellsto LAP and TGFβ activation by the same cells. See, FIGS. 2 and 3,respectively. Because the cells used also expressed the αvβ6 integrin,the results for these assays also include the β6 blocking antibody, 3G9.

Additional TGFβ activation studies were carried out with individualantibodies to determine their ability to block activation ofβ8-transfected SNB-19 cells at various concentrations. The results areshown in FIGS. 4-8 for ADWA-2, ADWA-11, ADWA-13, ADWA-15, and ADWA-16.Additional LAP blocking assays were also carried out in β8-transfectedSNB-19 cells. Results are shown in FIGS. 9-13.

The results show that ADWA-2, ADWA-16, ADWA-15, ADWA-11, ADWA-13, andADWA-10 are more effective for blocking LAP adhesion and inhibiting TGFβactivation.

Example 2

In order to further characterize the sequences of the ADWA-2, ADWA-8,ADWA-10, ADWA-11, ADWA-13, ADWA-15, ADWA-16, ADWA-20, and ADWA-25antibodies, total RNA was harvested from the hybridoma cell lineexpressing each antibody. RNA was isolated using Qiagen® RNeasy® kit andQIAshredder. Reverse transcriptase PCR was carried out using oligo dTand Clonetech® SMART™ IIa oligo. PCR was carried out using SMART-senseoligo and mouse IgG1 or kappa constant domain oligos.

Each of the 500 bp PCR products (from each antibody clone) was separatedusing gel electrophoresis, and cloned into Invitrogen™ Zero Blunt® TOPO®cloning kit, and transformed into cells. For each antibody hybridomaclone, 16 colonies were selected (288 total colonies).

The cloned DNA was sequenced by rolling circle amplification (RCA), andanalyzed with Invitrogen™ AlignX® and TIBCO® Spotfire®. The CDR andV-region sequences were initially analyzed to determine variabilitywithin each antibody clone, and then compared between clones.

Nearly all of the VH sequences of ADWA16 and ADWA2 were identical (SEQID NO:1), with 2 variants found in the 16 ADWA2 samples (SEQ ID NOs:2and 3). The primary ADWA13 sequence is shown in SEQ ID NO:4, with 2variants shown in SEQ ID NOs:5 and 6. The VH sequences of these threeantibodies was quite similar, with 2 amino acid changes in CDR2(positions 60 and 66) and 3 in the FW regions (positions 19, 45, and78).

The VH sequences for ADWA10, ADWA11, ADWA15, ADWA20, and ADWA25 wereidentical within each clone, but distinct from other clones. The VHsequences are shown in SEQ ID NOs: 7 (ADWA15), 8 (ADWA11), 9 (ADWA10),14 (ADWA20), and 15 (ADWA25). Three VH sequence variants were foundamong the 16 ADWA8 samples (SEQ ID NOs:11-13), with the primary sequenceshown as SEQ ID NO:10.

As for light chains, ADWA2, ADWA13, and ADWA16 were found to share thesame Vkappa sequence (SEQ ID NO:16). The Vkappa sequences for ADWA15,ADWA11, and ADWA10 are shown as SEQ ID NOs:17, 18, and 19, respectively.The Vkappa sequences for ADWA8, ADWA20, and ADWA25 are shown as SEQ IDNOs:20, 21, and 22, respectively.

The antibody sequences were confirmed and are shown in Table 1 below.Table 1 shows the sequences for the heavy chain variable (V_(H)) region,light chain variable (V_(L)) region, heavy chain CDRs (HCDR), lightchain CDRs (LCDR), V_(H)-encoding nucleotides, and V_(L)-encodingnucleotides. CDRs are shown according to both Chothia (underlined) andKabat (bold) designations.

TABLE 1  SEQ ID NO: Description Sequence V_(H) SEQUENCES-AMINO ACIDS 1ADWA2 QVQLQQSGAELAKPGASMKMSCKASGYTFSSY WIYWVKQRPGQGLEWIGYI N ADWA16PTTGY TEYNQKFRDKATLTADKSSNTAYMQLSSLTSEDSAVYYCAT EGGNWE DY WGQGTTLTVSS 2ADWA2-1 QVQLQQSGAELAKPGASMKMSCKASGYTFSSY WIYWVKQRPGQGLEWIGYI N PTTGYTEYNQKFRDKVTLTADKSSNTAYMQLSSLTSEDSAVYYCAT EGGNWE DY WGQGTTLTVSS 3ADWA2-2 QVQLQQSGAELTKPGASMKMSCKASGYTFSSY WIYWVKQRPGQGLEWIGYI N PTTGYTEYNQKFRDKVTLTADKSSNTAYMQLSSLTSEDSAVYYCAT EGGNWE DY WGQGTTLTVSS 4 ADWA13QVQLQQSGAELAKPGASVKMSCKASGYTFSSY WIYWVKQRPGQVLEWIGYI N PTTGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCAT EGGNWE DY WGQGTTLTVSS 5ADWA13-1 QVQLQQSGAELAKPGASVKMSCKASGYTFSSY WIYWVKQRPGQVLEWIGYI N PTTGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSTVYYCAT EGGNWE DY WGQGTTLTVSS 6ADWA13-2 QVQLQQSGAELTKPGASVKMSCKASGYTFSSY WIYWVKQRPGQVLEWIGYI N PTTGYTDYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSTVYYCAT EGGNWE DY WGQGTTLTVSS 7 ADWA15QVQLQQPGSVLVRPGASVKLSCKASGYTFTSS WMHWAKQRPGQGLEWIGEI H PNSGNSIYNEKFKDKATLTVDTSSSTAYVDLSSLTSEDSAVYYCAR WGDFDY WGQGTSVTVSS 8 ADWA11EVQLQQSGAELVRPGAFVKLSCKASGFNIKDY YMNWVLQRPEQGLEWIGWI D PDNGNTIYDPKFQGKASITADTSSNTAYLQLSSLTSEDTAVYYCAR RLLMDY WGQGTSVTVSS 9 ADWA10EVLLQQSGPELVKPGASVKIPCKASGYTFTNY NMDWVKQSHGKSLEWIGDI N PNSGGSVYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCAR WAYYGE RFHYFDY WGQGTTLTVSS 10ADWA8 QVQLQQPGSELVRPGASVKLSCKASGYTFTSY WMHWVKQRPGQGLEWIGNI Y PGSGRTNYDEKFKSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTR PLQYRY DVYPMDY WGQGTSVTVSS 11ADWA8-1 QVQLQQPGSELVRPGASVKLSCKASGYTFTSY WMHWVKQRPGQGLEWIGNI Y PGSGRTNYDEKFRSKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTR PLQYRY DVYPMDY WGQGTSVTVSS 12ADWA8-2 QVQLQQPGSELVRPGASVKLSCKASGYTFTSY WMHWVKQKPGQGLEWIGNI Y PGSGRTNYDEKFKSKATLTVDTSSSTAYMQLTSLTSEDSAVYYCTRPLQYRY DVYPMDY WGQGTSVTVSS 13ADWA8-3 QVQLQQPGSELVRPGASMKLSCKASGYTFTSY WMHWVKQRPGQGLEWIGNIY PGSGRTNYDEKFKSKATLTVDTSSSTAYMQLTSLTSEDSAVYYCTR PLQYRY DVYPMDY WGQGTSVTVSS 14ADWA20 DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDF AWSWIRQFPGNKLEWMGYI SYSGSTGYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCTR RGLYHW GFPY WGQGTLVTVSA 15ADWA25 EVQLVESGGDLVKPGGSLKLSCAASGFTESSY GMSWVRQTPDKRLEWVATI S GGGSYTYYPDSVKGRFTISRHNAKNTLYLQMSSLKSEDTAMYYCAS DPYYYG RRDLAWIAY WGQGTLVTVSAV_(L) SEQUENCES-AMINO ACIDS 16 ADWA2 DIQMTQTTSSLSASLGDRVTISC RASQDISNYLNWYQQKPDGTVKLLIY YTS ADWA13 RLYS GVPSRFSGSGSGTDYSLTISNLEPKDIATYYCQQFSELPRT EGGGTKL ADWA16 EIK 17 ADWA15 DVQMTQTTSSLSASLGDRVTISCRASQDISNYLN WYQQKPDGTVKLLIY YTS RLHS GVPSRFSGSGSGTDFSLTISNLEPEDIATYYCQQYNKVPLT FGGGTKL ELK 18 ADWA11 DIVMTQAAPSVPVTPGESVSISC RSTKSLLHENGNTYLFWFLQRPGQSPQRL IY YMSNLAS GVPDRFSGRGSGTDFTLRISRVEAEDVGVYYC MQSLEYPFT FGTGTKLEIK 19 ADWA10 QIVLSQSPAILSASPGEKVTMTC RASSSVSYMH WYQQKSGSSPKPWIYATSN LAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYC QQWSSNPPT FGGGTKLE IK 20 ADWA8DIQMTQSPASLSASVGETVTITC RASENIDSYLA WYQQKQGKSPQLLVY AAT LLPDGVPSRFSGSGSGTQYSLKINSLQSEDVARYYC QHYYNTPWT FGGGTKL EIK 21 ADWA20DIVMTQSHKFMSTSVGDRVSITC KASQDVSSAVA WYQQKPGQSPKLLIY WAS TRHTGVPDRFTGSGSGTDYTLTVSNVQAEDLALYYC QQHYITPYT FGGGTKL EIK 22 ADWA25DIVMTQSQKFMSTSVGDRVSVTC KASQNVGTNVA WYQQKPGQSPKALIY SAS YRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC QQYNSYPYT EGGGTKL EIKV_(H) CDR SEQUENCES-AMINO ACIDS 23 ADWA2 GYTFSSY WIY (UNDERLINE =SEQ ID NO: 88; BOLD = SEQ ID NO: 89) ADWA16 ADWA2-1 ADWA2-2 ADWA13ADWA13-1 ADWA13-2 HCDR1 24 ADWA2 YI NPTTGY TEYNQKFRD (UNDERLINE =SEQ ID NO: 90) ADWA16 ADWA2-1 ADWA2-2 HCDR2 25 ADWA2 EGGNWEDY ADWA16ADWA2-1 ADWA2-2 ADWA13 ADWA13-1 ADWA13-2 HCDR3 26 ADWA13 YI NPTTGYTDYNQKFKD (UNDERLINE = SEQ ID NO: 90) ADWA13-1 ADWA13-2 HCDR2 27 ADWA15GYTFTSS WMH (UNDERLINE = SEQ ID NO: 91; BOLD = SEQ ID NO: 92) HCDR1 28ADWA15 EI HPNSGN SIYNEKFKD (UNDERLINE = SEQ ID NO: 93) HCDR2 29 ADWA15WGDFDY HCDR3 30 ADWA11 GFNIKDY YMN (UNDERLINE = SEQ ID NO: 94; BOLD =SEQ ID NO: 95) HCDR1 31 ADWA11 WI DPDNGN TIYDPKFQG (UNDERLINE =SEQ ID NO: 96) HCDR2 32 ADWA11 RLLMDY HCDR3 33 ADWA10 GYTFTNYNMD (UNDERLINE = SEQ ID NO: 97; BOLD = SEQ ID NO: 98) HCDR1 34 ADWA10 DINPNSGG SVYNQKFKG (UNDERLINE = SEQ ID NO: 99) HCDR2 35 ADWA10WAYYGERFHYFDY HCDR3 36 ADWA8 GYTFTSY WMH (UNDERLINE =SEQ ID NO: 100; BOLD = SEQ ID NO: 101) ADWA8-1 ADWA8-2 ADWA8-3 HCDR1 37ADWA8 NI YPGSGR TNYDEKFKS (UNDERLINE = SEQ ID NO: 102) ADWA8-2 ADWA8-3HCDR2 38 ADWA8 PLQYRYDVYPMDY ADWA8-1 ADWA8-2 ADWA8-3 HCDR3 39 ADWA8-1 NIYPGSGR TNYDEKFRS (UNDERLINE = SEQ ID NO: 102) HCDR2 40 ADWA20 GYSITSDFAWS (UNDERLINE = SEQ ID NO: 103; BOLD = SEQ ID NO: 104) HCDR1 41 ADWA20YI SYSGS TGYNPSLKS (UNDERLINE = SEQ ID NO: 105) HCDR2 42 ADWA20RGLYHWGFPY HCDR3 43 ADWA25 GFTFSSY GMS (UNDERLINE =SEQ ID NO: 106; BOLD = SEQ ID NO: 107) HCDR1 44 ADWA25 TI SGGGSYTYYPDSVKG HCDR2 45 ADWA25 DPYYYGRRDLAWIAY HCDR3V_(L) CDR SEQUENCES-AMINO ACIDS 46  ADWA2 RASQDISNYLN ADWA13 ADWA16ADWA15 LCDR1 47 ADWA2 YTSRLYS ADWA13 ADWA16 LCDR2 48 ADWA2 QQFSELPRTADWA13 ADWA16 LCDR3 49 ADWA15 YTSRLHS LCDR2 50 ADWA15 QQYNKVPLT LCDR3 51ADWA11 RSTKSLLHFNGNTYLF LCDR1 52 ADWA11 YMSNLAS LCDR2 53 ADWA11MQSLEYPFT LCDR3 54 ADWA10 RASSSVSYMH LCDR1 55 ADWA10 ATSNLAS LCDR2 56ADWA10 QQWSSNPPT LCDR3 57 ADWA8 RASENIDSYLA LCDR1 58 ADWA8 AATLLPD LCDR259 ADWA8 QHYYNTPWT LCDR3 60 ADWA20 KASQDVSSAVA LCDR1 61 ADWA20 WASTRHTLCDR2 62 ADWA20 QQHYITPYT LCDR3 63 ADWA25 KASQNVGTNVA LCDR1 64 ADWA25SASYRYS LCDR2 65 ADWA25 QQYNSYPYT LCDR3 V_(H) SEQUENCES-NUCLEIC ACIDS 66ADWA16-1 CAGGTCCAGCTTCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAATGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTTCTAGCTACTGGATATATTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTACCACTGGTTATACTGAGTACAATCAGAAGTTCAGGGACAAGGCCACATTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAACAGAGGGAGGTAATTGGGAGGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 67 ADWA2-1CAGGTCCAGCTTCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAATGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTTCTAGCTACTGGATATATTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTACCACTGGTTATACTGAGTACAATCAGAAGTTCAGGGACAAGGTCACATTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAACAGAGGGAGGTAATTGGGAGGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 68 ADWA2-2CAGGTCCAGCTTCAGCAGTCTGGGGCTGAACTGACAAAACCTGGGGCCTCAATGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTTCTAGCTACTGGATATATTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTACCACTGGTTATACTGAGTACAATCAGAAGTTCAGGGACAAGGCCACATTGACTGCAGACAAATCCTCCAACACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAACAGAGGGAGGTAATTGGGAGGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 69 ADWA13CAGGTCCAGCTTCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTTCTAGCTACTGGATATATTGGGTAAAACAGAGGCCTGGACAGGTTCTGGAATGGATTGGATACATTAATCCTACCACTGGTTACACTGACTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAACAGAGGGAGGTAATTGGGAGGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 70 ADWA13-1CAGGTCCAGCTTCAGCAGTCTGGGGCTGAACTGGCAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTTCTAGCTACTGGATATATTGGGTAAAACAGAGGCCTGGACAGGTTCTGGAATGGATTGGATACATTAATCCTACCACTGGTTACACTGACTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTACAGTCTATTACTGTGCAACAGAGGGAGGTAATTGGGAGGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 71 ADWA13-2CAGGTCCAGCTTCAGCAGTCTGGGGCTGAACTGACAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTTCTAGCTACTGGATATATTGGGTAAAACAGAGGCCTGGACAGGTTCTGGAATGGATTGGATACATTAATCCTACCACTGGTTACACTGACTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAACAGAGGGAGGTAATTGGGAGGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 72 ADWA15CAGGTCCAACTGCAGCAGCCTGGGTCTGTGCTGGTGAGGCCTGGAGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTCCTGGATGCACTGGGCGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTCATCCTAATAGTGGTAATAGTATCTACAATGAGAAGTTCAAGGACAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACGTGGATCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATGGGGGGATTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA 73 ADWA11GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCTGGGGCCTTTGTCAAGTTGTCCTGCAAGGCTTCTGGCTTCAACATTAAAGACTACTATATGAATTGGGTGTTGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGACAATGGTAATACTATATATGACCCGAAGTTCCAGGGCAAGGCCAGTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGAAGACTACTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 74 ADWA10GAGGTCCTGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATACCCTGCAAGGCTTCTGGATACACATTCACTAACTACAACATGGACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGATATTAATCCTAACAGTGGTGGTTCTGTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGATGGGCCTACTATGGTGAAAGGTTTCACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCT CA 75 ADWA8CAGGTCCAACTGCAGCAACCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCAGGCTACACATTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTTATCCTGGTAGTGGTAGAACTAACTACGACGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGACCGCTCCAGTATAGGTACGACGTCTATCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCT CA 76 ADWA8-1CAGGTCCAACTGCAGCAACCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCAGGCTACACATTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTTATCCTGGTAGTGGTAGAACTAACTACGACGAGAAGTTCAGGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGACCGCTCCAGTATAGGTACGACGTCTATCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCT CA 77 ADWA8-2CAGGTCCAACTGCAGCAACCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAATGAAGCTGTCCTGCAAGGCTTCAGGCTACACATTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTTATCCTGGTAGTGGTAGAACTAACTACGACGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAACTCACCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGACCGCTCCAGTATAGGTACGACGTCTATCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCT CA 78 ADWA8-3CAGGTCCAACTGCAGCAACCTGGGTCTGAGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCAGGCTACACATTCACCAGCTACTGGATGCACTGGGTGAAGCAGAAGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTTATCCTGGTAGTGGTAGAACTAACTACGACGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAACTCACCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGACCGCTCCAGTATAGGTACGACGTCTATCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCT CA 79 ADWA20GATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTTTGCCTGGAGCTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAGCTACAGTGGTAGCACTGGCTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTACAAGAAGGGGCCTCTACCACTGGGGGTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA 80 ADWA25GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTGGTGGTGGTAGTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGACACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGCGACCCCTATTACTACGGTAGAAGGGACCTGGCCTGGATTGCTTACTGGGGCCAAGGGACTCTGGTCACTG TCTCTGCAV_(L) SEQUENCES-NUCLEIC ACIDS 81 ADWA2GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACA ADWA13GAGTCACCATCAGTTGCAGGGCAAGTCAGGATATTAGCAATTATTTAAACTG ADWA16GTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCTAGATTATACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTAAAGATATTGCCACTTACTATTGTCAGCAGTTTAGTGAGCTTCCTCGGACGTTCGGTGGAGGCACCAAGCTG GAAATCAAA 82 ADWA15GATGTCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGTAGGGCAAGTCAGGATATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCACGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTTTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAATACAATAAGGTTCCGCTCACGTTCGGTGGTGGGACCAAGCTG GAGCTGAAA 83 ADWA11GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTTTCCATCTCCTGCAGGTCTACTAAGAGTCTTCTGCATTTTAATGGCAACACTTACTTGTTTTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCGCCTGATATATTATATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGAGGGTCAGGAACTGATTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAAAGTCTAGAATATCCATTCACGTTCGGCACGGGGACAAAATTGGAAATAAAA 84 ADWA10AGGTCACAATGACTTGCAGGGCCAGTTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAA ATAAAA 85 ADWA8GACATCCAGATGACTCAGTCTCCAGCTTCCCTGTCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTGACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGCTGCAACACTCTTACCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCTCTCAAGATCAACAGCCTGCAGTCTGAAGATGTTGCGAGATATTACTGTCAACATTATTATAATACTCCGTGGACGTTCGGTGGAGGCACCAAGCTG GAAATCAAA 86 ADWA20GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTGGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTAGTGCTGTAGCCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTCCTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCACCGTCAGCAATGTGCAGGCTGAAGACCTGGCACTTTATTACTGTCAGCAACATTATATCACTCCTTACACGTTCGGAGGGGGGACCAAGCTG GAAATAAAA 87 ADWA25GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTG GAAATAAAA

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, internet sources, patents,patent applications, and accession numbers cited herein are herebyincorporated by reference in their entireties for all purposes.

What is claimed is:
 1. An isolated antibody that specifically binds tohuman integrin β8 and inhibits adhesion of latency associated peptide(LAP) to αvβ8, wherein the antibody comprises: a heavy chain variableregion comprising the Kabat-determined or Chothia-determinedcomplementarity determining regions (HCDR1, HCDR2, and HCDR3) of SEQ IDNO:1 and a light chain variable region comprising the Kabat-determinedor Chothia-determined complementarity determining regions (LCDR1, LCDR2,and LCDR3) of SEQ ID NO:16; or a heavy chain variable region comprisingthe Kabat-determined or Chothia-determined complementarity determiningregions (HCDR1, HCDR2, and HCDR3) of SEQ ID NO:2 and a light chainvariable region comprising the Kabat-determined or Chothia-determinedcomplementarity determining regions (LCDR1, LCDR2, and LCDR3) of SEQ IDNO:16; or a heavy chain variable region comprising the Kabat-determinedor Chothia-determined complementarity determining regions (HCDR1, HCDR2,and HCDR3) of SEQ ID NO:3 and a light chain variable region comprisingthe Kabat-determined or Chothia-determined complementarity determiningregions (LCDR1, LCDR2, and LCDR3) of SEQ ID NO:16; or a heavy chainvariable region comprising the Kabat-determined or Chothia-determinedcomplementarity determining regions (HCDR1, HCDR2, and HCDR3) of SEQ IDNO:4 and a light chain variable region comprising the Kabat-determinedor Chothia-determined complementarity determining regions (LCDR1, LCDR2,and LCDR3) of SEQ ID NO:16; or a heavy chain variable region comprisingthe Kabat-determined or Chothia-determined complementarity determiningregions (HCDR1, HCDR2, and HCDR3) of SEQ ID NO:5 and a light chainvariable region comprising the Kabat-determined or Chothia-determinedcomplementarity determining regions (LCDR1, LCDR2, and LCDR3) of SEQ IDNO:16; or a heavy chain variable region comprising the Kabat-determinedor Chothia-determined complementarity determining regions (HCDR1, HCDR2,and HCDR3) of SEQ ID NO:6 and a light chain variable region comprisingthe Kabat-determined or Chothia-determined complementarity determiningregions (LCDR1, LCDR2, and LCDR3) of SEQ ID NO:16; or a heavy chainvariable region comprising the Kabat-determined or Chothia-determinedcomplementarity determining regions (HCDR1, HCDR2, and HCDR3) of SEQ IDNO:7 and a light chain variable region comprising the Kabat-determinedor Chothia-determined complementarity determining regions (LCDR1, LCDR2,and LCDR3) of SEQ ID NO:17; or a heavy chain variable region comprisingthe Kabat-determined or Chothia-determined complementarity determiningregions (HCDR1, HCDR2, and HCDR3) of SEQ ID NO:8 and a light chainvariable region comprising the Kabat-determined or Chothia-determinedcomplementarity determining regions (LCDR1, LCDR2, and LCDR3) of SEQ IDNO:18; or a heavy chain variable region comprising the Kabat-determinedor Chothia-determined complementarity determining regions (HCDR1, HCDR2,and HCDR3) of SEQ ID NO:9 and a light chain variable region comprisingthe Kabat-determined or Chothia-determined complementarity determiningregions (LCDR1, LCDR2, and LCDR3) of SEQ ID NO:19; or a heavy chainvariable region comprising the Kabat-determined or Chothia-determinedcomplementarity determining regions (HCDR1, HCDR2, and HCDR3) of SEQ IDNO:10 and a light chain variable region comprising the Kabat-determinedor Chothia-determined complementarity determining regions (LCDR1, LCDR2,and LCDR3) of SEQ ID NO:20; or a heavy chain variable region comprisingthe Kabat-determined or Chothia-determined complementarity determiningregions (HCDR1, HCDR2, and HCDR3) of SEQ ID NO:11 and a light chainvariable region comprising the Kabat-determined or Chothia-determinedcomplementarity determining regions (LCDR1, LCDR2, and LCDR3) of SEQ IDNO:20; or a heavy chain variable region comprising the Kabat-determinedor Chothia-determined complementarity determining regions (HCDR1, HCDR2,and HCDR3) of SEQ ID NO:12 and a light chain variable region comprisingthe Kabat-determined or Chothia-determined complementarity determiningregions (LCDR1, LCDR2, and LCDR3) of SEQ ID NO:20; or a heavy chainvariable region comprising the Kabat-determined or Chothia-determinedcomplementarity determining regions (HCDR1, HCDR2, and HCDR3) of SEQ IDNO:13 and a light chain variable region comprising the Kabat-determinedor Chothia-determined complementarity determining regions (LCDR1, LCDR2,and LCDR3) of SEQ ID NO:20; or a heavy chain variable region comprisingthe Kabat-determined or Chothia-determined complementarity determiningregions (HCDR1, HCDR2, and HCDR3) of SEQ ID NO:14 and a light chainvariable region comprising the Kabat-determined or Chothia-determinedcomplementarity determining regions (LCDR1, LCDR2, and LCDR3) of SEQ IDNO:21, or a heavy chain variable region comprising the Kabat-determinedor Chothia-determined complementarity determining regions (HCDR1, HCDR2,and HCDR3) of SEQ ID NO:15 and a light chain variable region comprisingthe complementarity determining regions (LCDR1, LCDR2, and LCDR3) of SEQID NO:22.
 2. The antibody of claim 1, wherein the antibody comprises oneor more human framework regions.
 3. A pharmaceutical compositioncomprising the antibody of claim
 1. 4. The isolated antibody of claim 1,wherein HCDR1 comprises SEQ ID NO:88, HCDR2 comprises SEQ ID NO:90, andHCDR3 comprises SEQ ID NO:25, and LCDR1 comprises SEQ ID NO:46, LCDR2comprises SEQ ID NO:47, and LCDR3 comprises SEQ ID NO:48.
 5. Theisolated antibody of claim 1, wherein HCDR1 comprises SEQ ID NO:89,HCDR2 comprises SEQ ID NO:24, and HCDR3 comprises SEQ ID NO:25, andLCDR1 comprises SEQ ID NO:46, LCDR2 comprises SEQ ID NO:47, and LCDR3comprises SEQ ID NO:48.
 6. The isolated antibody of claim 1, whereinHCDR1 comprises SEQ ID NO:88, HCDR2 comprises SEQ ID NO:90, and HCDR3comprises SEQ ID NO:25, and LCDR1 comprises SEQ ID NO:46, LCDR2comprises SEQ ID NO:47, and LCDR3 comprises SEQ ID NO:48.
 7. Theisolated antibody of claim 1, wherein HCDR1 comprises SEQ ID NO:89,HCDR2 comprises SEQ ID NO:26, and HCDR3 comprises SEQ ID NO:25, andLCDR1 comprises SEQ ID NO:46, LCDR2 comprises SEQ ID NO:47, and LCDR3comprises SEQ ID NO:48.
 8. The isolated antibody of claim 1, whereinHCDR1 comprises SEQ ID NO:91, HCDR2 comprises SEQ ID NO:93, and HCDR3comprises SEQ ID NO:29, and LCDR1 comprises SEQ ID NO:46, LCDR2comprises SEQ ID NO:49, and LCDR3 comprises SEQ ID NO:50.
 9. Theisolated antibody of claim 1, wherein HCDR1 comprises SEQ ID NO:92,HCDR2 comprises SEQ ID NO:28, and HCDR3 comprises SEQ ID NO:29, andLCDR1 comprises SEQ ID NO:46, LCDR2 comprises SEQ ID NO:49, and LCDR3comprises SEQ ID NO:50.
 10. The isolated antibody of claim 1, whereinHCDR1 comprises SEQ ID NO:94, HCDR2 comprises SEQ ID NO:96, and HCDR3comprises SEQ ID NO:32, and LCDR1 comprises SEQ ID NO:51, LCDR2comprises SEQ ID NO:52, and LCDR3 comprises SEQ ID NO:53.
 11. Theisolated antibody of claim 1, wherein HCDR1 comprises SEQ ID NO:95,HCDR2 comprises SEQ ID NO:31, and HCDR3 comprises SEQ ID NO:32, andLCDR1 comprises SEQ ID NO:51, LCDR2 comprises SEQ ID NO:52, and LCDR3comprises SEQ ID NO:53.
 12. The isolated antibody of claim 1, whereinHCDR1 comprises SEQ ID NO:97, HCDR2 comprises SEQ ID NO:99, and HCDR3comprises SEQ ID NO:35, and LCDR1 comprises SEQ ID NO:54, LCDR2comprises SEQ ID NO:55, and LCDR3 comprises SEQ ID NO:56.
 13. Theisolated antibody of claim 1, wherein HCDR1 comprises SEQ ID NO:98,HCDR2 comprises SEQ ID NO:34, and HCDR3 comprises SEQ ID NO:35, andLCDR1 comprises SEQ ID NO:54, LCDR2 comprises SEQ ID NO:55, and LCDR3comprises SEQ ID NO:56.
 14. The isolated antibody of claim 1, whereinHCDR1 comprises SEQ ID NO:100, HCDR2 comprises SEQ ID NO:102, and HCDR3comprises SEQ ID NO:38, and LCDR1 comprises SEQ ID NO:57, LCDR2comprises SEQ ID NO:58, and LCDR3 comprises SEQ ID NO:59.
 15. Theisolated antibody of claim 1, wherein HCDR1 comprises SEQ ID NO:101,HCDR2 comprises SEQ ID NO:37, and HCDR3 comprises SEQ ID NO:38, andLCDR1 comprises SEQ ID NO:57, LCDR2 comprises SEQ ID NO:58, and LCDR3comprises SEQ ID NO:59.
 16. The isolated antibody of claim 1, whereinHCDR1 comprises SEQ ID NO:101, HCDR2 comprises SEQ ID NO:39, and HCDR3comprises SEQ ID NO:38, and LCDR1 comprises SEQ ID NO:57, LCDR2comprises SEQ ID NO:58, and LCDR3 comprises SEQ ID NO:59.
 17. Theisolated antibody of claim 1, wherein HCDR1 comprises SEQ ID NO:103,HCDR2 comprises SEQ ID NO:105, and HCDR3 comprises SEQ ID NO:42, andLCDR1 comprises SEQ ID NO:60, LCDR2 comprises SEQ ID NO:61, and LCDR3comprises SEQ ID NO:62.
 18. The isolated antibody of claim 1, whereinHCDR1 comprises SEQ ID NO:104, HCDR2 comprises SEQ ID NO:41, and HCDR3comprises SEQ ID NO:42, and LCDR1 comprises SEQ ID NO:60, LCDR2comprises SEQ ID NO:61, and LCDR3 comprises SEQ ID NO:62.
 19. Theisolated antibody of claim 1, wherein HCDR1 comprises SEQ ID NO:106,HCDR2 comprises SEQ ID NO:108, and HCDR3 comprises SEQ ID NO:45, andLCDR1 comprises SEQ ID NO:63, LCDR2 comprises SEQ ID NO:64, and LCDR3comprises SEQ ID NO:65.
 20. The isolated antibody of claim 1, whereinHCDR1 comprises SEQ ID NO:107, HCDR2 comprises SEQ ID NO:44, and HCDR3comprises SEQ ID NO:45, and LCDR1 comprises SEQ ID NO:63, LCDR2comprises SEQ ID NO:64, and LCDR3 comprises SEQ ID NO:65.